A serum protein specific to the fetus was first demonstrated by Pederson, Nature (London) 154, 575 (1944) in calf serum and by Bergstand et al. Scand. J. Clin. Lab. Invest 8 174 (1956) in humans. This feto-specific protein was designated alpha.sub.1 -fetoprotein (AFP) due to its electrophoretic mobility. Alpha.sub.1 -fetoprotein in sera of hepatocellular cancer and malignant embryonal teratoma patients was found to be immunologically and electrophoretically identical with alpha.sub.1 -fetoprotein observed in fetal serum by Tatarinov, Vopr. Med. Khim. 11 20-24 (1965) and Abelev et al., Int. J. Cancer 2 551-558 (1967).
Human alpha.sub.1 -fetoprotein has been isolated from fetal serum by chemical procedures by Pederson, Clin. Chimica Acta 38 163-170 (1971), Ruoslahit et al., Int. J. Cancer 7 218-225 (1971), Silver et al., Proc. Nat. Acad. Sci., U.S.A. 70 526-530 (1973), Forrester et al., Clin. Chimica Acta 64 317-323 (1975) and Twomey et al., Clin. Chem. 22 1306-1309 (1976). In addition immuno-chemical and chemical methods have been employed to isolate alpha.sub.1 -fetoprotein from hepatoma serum by Nishi, Cancer Res. 30 2507-2513 (1970), Nishi et al., Protides Biol. Fluids 18 43-47 (1971), Nishi et al., Biochem. Biophys. Acta 278 293-298 (1972), Aoyagi et al., Cancer Research 37 3663-3667 (Oct. 1977), Yachnin et al., Biochem. Biophys. Acta 493 418-428 (1977), Lehmann et al., Clin. Chimica Acta 33 197-206 (1971) and Alpert et al., J. Biol. Chem 247 3492-3497 (1972).
The methods referred to above result in an alpha.sub.1 -fetoprotein (AFP) which is not pure in that it contains albumin and other proteins. Because of the presence of these contaminants, such relatively impure alpha.sub.1 -fetoprotein preparations are not satisfactory as the radiolabeled antigen for a sensitive radioimmunoassay. Further, such preparations are not satisfactory for the production of monospecific high titer antisera for a sensitive radioimmunoassay (RIA). Because samples to be tested for AFP in a radioimmunoassy often contain AFP in low concentration, it is essential that the RIA procedure be highly sensitive. By highly sensitive is meants that the RIA must be able to accurately detect AFP at a level of about 20 ng/ml of sample. Such sensitive RIA procedures are required for screening for birth defects in pregnant women, which has heretofore not been feasible.
Non-specific antisera or impure labeled AFP cannot be utilized in such sensitive RIA procedures since they would not yield an accurate measure of the AFP content of the sample. In addition, the methods of alpha.sub.1 -fetoprotein reported in the literature are not satisfactory for isolating AFP from large volumes of source solutions and/or source solutions which contain low concentrations of AFP. Further, the methods disclosed in the literature for isolating AFP do not appear to be satisfactory for automation techniques.
Specifically, the Ruoslahti et al. article teaches a method for purifying AFP from fetal serum utilizing two electrofocusing procedures in series or immunoprecipitation. The electrofocusing procedures are basically separations based on charge. There is no disclosure that the AFP obtained by the disclosed procedure would be of sufficient purity to be utilized in a RIA.
Alpert et al. teach a method of purifying AFP by a three step chemical process including: (1) starch gel block electrophoresis, (2) gel filtration utilizing a Sephadex column, and (3) isoelectric focusing. The AFP thus obtained is stated to be pure by immunodiffusion and sodium dodecyl sulfate gel electrophoresis. In addition, the AFP thus obtained is stated as being capable of producing immunospecific antiserum in rabbits. In contrast to the method of the captioned application which utilizes immunological as well as chemical techniques, there is no suggestion that the AFP produced by the disclosed method is sufficiently pure for RIA procedures.
The first two Nishi articles listed above utilize hepatoma serum as source material and purify basically by immunoprecipitation followed by gel filtration.
The method of the third Nishi article also utilizes hepatoma serum as a source material and teaches a purification method utilizing an immunosorbent column. However, the antibody is not adsorbed to insure monospecificity. Further purification is carried out by gel filtration.
None of the methods disclosed in the Nishi articles appears to teach one skilled in the art how to purify AFP to a degree sufficient for use in RIA procedures. Certainly, none contains even the slightest suggestion that AFP can be obtained from monkey source material in sufficient purity for use in human RIA procedures.
Aoyagi et al. teach a method of purifying AFP from cord serum utilizing an immunosorbent column, ion exchange on DEAE Sephadex and gel filtration. Again, there is no indication of the AFP purified by this procedure being suitable for RIA procedures. Although ion exchange and gel filtration chromatography does not afford optimum separation of albumin due to closeness of charge and molecular weight, the AFP produced by the disclosed procedure appears to be relatively pure. There is no suggestion, however, that the disclosed method could produce AFP suitable for human RIAs from monkey source material in contrast to the method of the present invention.
Yachnin et al. disclose a method of purifying AFP hapatoma serum utilizing an immunosorbent column and gel filtration. It appears from the disclosure that the product is microheterogeneous and, therefore, unsatisfactory for RIA procedures.
There is thus a need for a method of isolating alpha.sub.1 -fetoprotein in as pure a state as possible in terms of albumin and other protein contaminants which would interfere with sensitive radioimmunoassays. Further, there is a need for isolating AFP which can be efficiently applied to large volumes of source solutions and/or source solutions which contain AFP in low concentration. Finally, there is a need for a method of isolating AFP which is amenable to being carried out, wholly or partially, by automated apparatus and techniques. These needs are all satisfied by the method of the subject invention. Further, the methodology of the present invention affords a means whereby AFP can be obtained from monkey hepatoma serum in sufficient purity so as to be for practical purposes, the same as that obtained from human cord serum and sufficiently pure to be used in RIA procedures on human samples. Monkey AFP has heretofore not been recognized as being usable in radioimmunoassays on human material.